Methods of modulating fibrosis

ABSTRACT

The invention features methods of modulating fibrosis and/or angiogenesis by inhibiting components of the VEGF signaling pathway. The methods are useful in the treatment of fibrotic and or angiogenesis related disorders.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisionalapplication No. 60/219,244, filed on Jul. 18, 2000, the contents ofwhich are incorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The U.S. Government may have certain rights in this inventionpursuant to Grant No. EY5110 awarded by the National Institutes ofHealth to George L. King.

BACKGROUND

[0003] Angiogenesis and fibrosis are key components in development,growth, wound healing, and regeneration (Klagsbrun & D'Amore, (1991)Annu. Rev. Physiol. 53, 217-239). In addition, these processes commonlyoccur together in many disease states where neovascularization isbelieved to initiate the pathological cascade, including proliferativediabetic retinopathy (Aiello et al. (1998) Diabetes Care 21, 143-156),rheumatoid arthritis (Firestein (1999) J. Clin. Invest. 103, 3-4), andage-related macular degeneration (Lopez et al. (1996) Invest.Ophthalmol. & Visual Sci. 37, 855-868).

[0004] Vascular endothelial growth factor (VEGF) is expressed as afamily of peptides of 121, 145, 165, 189, and 206 amino acid residues.Its expression is induced by hypoxia and is essential in thevasculogenesis process during development. Several receptors have beenshown to mediate the action of VEGF, and most of them belong to thetyrosine kinase receptor family (Petrova et al. (1999) Exp. Cell Res.253, 117-130). Upon the binding of VEGF to its receptors, multiplesignaling cascades are activated, including the tyrosine phosphorylationof phospholipase Cγ, elevation of intracellular calcium anddiacylglycerol, activation of protein kinase C (PKC), and extracellularsignal-regulated kinase (MAPK/ERK) for endothelial cell proliferation.In addition, VEGF also stimulates activation of phosphatidylinositol(PI) 3-kinase leading to Akt/PKB activation and possibly enhancingendothelial cell survival.

[0005] Connective tissue growth factor (CTGF) is a potent andubiquitously expressed growth factor that has been shown to play aunique role in fibroblast proliferation, cell adhesion, and thestimulation of extracellular matrix production (Frazier et al. (1996) J.Invest. Dermatol. 107, 404-411; Kireeva et al. (1997) Exp. Cell Res.233, 63-77). The 38-kDa protein was originally identified in conditionedmedium from human umbilical vein endothelial cells, and the expressionwas shown to be selectively stimulated by transforming growth factor-β(TGF-β) in cultured fibroblasts. Due to its mitogenic action onfibroblasts and its ability to induce the expression of theextracellular matrix molecules, collagen type I, fibronectin, andintegrin α5, CTGF is supposed to play an important role in connectivetissue cell proliferation and extracellular matrix deposition as one ofthe mediators of TGF-β. CTGF also seems to be an important player in thepathogenesis of various fibrotic disorders, since it was shown to beoverexpressed in scleroderma, keloids, and other fibrotic skin disorders(Igarashi et al. (1996) J. Invest. Dermatol. 106, 729-733), as well asin stromal rich mammary tumors, and in advanced atherosclerotic lesions.Recently, the integrin α5β3 has been reported to serve as a receptor onendothelial cells for CTGF-mediated endothelial cell adhesion,migration, and angiogenesis (Babic et al. (1999) Mol. Cell. Biol. 19,2958-2966).

[0006] Besides TGF-β, the expression of CTGF is reported to be regulatedby dexamethasone in BALB/c 3T3 cells, high glucose in human mesangialcells, kinin in human embryonic fibroblasts, factor VIIa, and thrombinin WI-38 fibroblasts, tumor necrosis factor α in human skin fibroblast,and cAMP in bovine endothelial cells (Dammeier et al. (1998) J. Biol.Chem. 273, 18185-18190; Murphy et al. (1999) J. Biol. Chem. 274,5830-5834; Ricupero et al. (2000) J. Biol. Chem. 275, 12475-12480;Pendurthi et al. (2000) J. Biol. Chem. 275, 14632-14641; Abraham et al.(2000) J. Biol. Chem. 275, 15220-15225; Boes et al. (1999) Endocrinology140, 1575-1580).

SUMMARY

[0007] The invention is based, in part, on the discovery that VEGF canregulate connective tissue growth factor (CTGF), e.g., through the PI3Kinase-Akt pathway. CTGF is a potent diffusible growth factor whichregulates extracellular matrix deposition and connective tissue cellproliferation. CTGF is a potent activator of fibrosis, angiogenesis, andextracellular matrix production. Modulation of the levels of CTGF viathe VEGF pathway, e.g., by modulation of a VEGF activity, e.g., a VEGFsignaling activity, e.g., a VEGF receptor (VEGFR) activity, e.g.,KDR/flk1, Flt1, Flt 4, neuropilin-1 (NP-1), tie, or tie2 activity;modulation of P13-kinase; or modulation of Akt can thereby stimulate oralternatively reduce fibrosis and/or angiogenesis. Accordingly, oneaspect of the invention features methods of modulating fibrosis. Anotheraspect features methods of modulating angiogenesis.

[0008] In one aspect, the invention features a method of decreasingfibrosis in a tissue, e.g., a skin, lung, retinal, renal, cardiac, orliver tissue of a subject, e.g., a human or non-human animal, bydecreasing CTGF activity or expression. CTGF activity or expression isdecreased by decreasing a VEGF signaling activity. In a preferredembodiment, the method includes identifying a subject in need ofdecreased fibrosis, e.g., a subject having a fibrotic disorder, e.g., adisorder described herein.

[0009] In a preferred embodiment, VEGF signaling is decreased byadministering an agent that inhibits a component of the VEGF signaltransduction pathway, e.g., an agent that decreases VEGF activity,decreases VEGF receptor (VEGFR) activity, e.g., KDR/flk1, Flt1, Flt4,NP-1, tie, or tie2 activity; decreases PI3-kinase activity; decreases anAkt activity; decreases an Erk activity.

[0010] In a preferred embodiment, KDR, Flt-4, Flt-1 or neuropilinactivity is decreased.

[0011] In a preferred embodiment, the subject has a disorder related tounwanted or excessive fibrosis. In some embodiments, fibrosis isexhibited in, e.g., skin, liver, kidney, cardiac, or lung tissue. Thedisorder can be caused by scarring, e.g., keloid formation. Examples ofother disorders related to unwanted or excessive fibrosis include, butare not limited to, scleroderma (e.g., morphea, generalized morphea,linear scleroderma); keloids; kidney fibrosis, e.g., glomerularsclerosis or renal tubulointerstitial fibrosis; pulmonary fibrosis,e.g., diffuse interstitial pulmonary fibrosis; cardiac fibrosis;chemotherapy/radiation induced lung fibrosis; pancreatitis; a disease ofthe kidney, e.g., glomerular sclerosis, renal tubulointerstitialfibrosis, or progressive renal disease; atherosclerotic plaques, e.g.,restenosis; inflammatory bowel disease; Crohn's disease; arthriticjoints, e.g., rheumatoid arthritis; cancer, e.g., invasive breastcarcinoma, stromal rich mammary tumors, dermatofibromas, angiolipoma,and angioleiomyoma; hypertrophic scar; nodular fasciitis, eosinophilicfasciitis, dupuytren's contracture; macular degeneration, e.g.,age-related macular degeneration; acute ocular neovascularization ordiabetic retinopathy; general fibrosis syndrome, characterized byreplacement of normal muscle tissue by fibrous tissue in varyingdegrees; retroperitoneal fibrosis; liver fibrosis; acute fibrosis, e.g.,in response to various forms of trauma including accidental injuries,infections, surgery, burns, radiation or chemotherapy treatments; ormacular degeneration, e.g., age-related macular degeneration;

[0012] The method includes administering an agent which decreases CTGFactivity or expression. In a preferred embodiment, the agent is an agentdescribed herein that decreases a VEGF signaling activity.

[0013] In a preferred embodiment, CTGF activity or expression isdecreased in an endothelial cell.

[0014] In a preferred embodiment, CTGF activity or expression isdecreased in a pericyte.

[0015] An agent which decreases CTGF activity can be one or more of: anagent which decreases the level or activity of VEGF, e.g., an agentwhich inhibits VEGF interaction with a VEGF receptor (VEGFR), e.g.,flt4, flt1, NP-1 tie, tie-2, and/or KDR/flk1; an agent which inhibitsVEGF receptor activation; an agent which disrupts a VEGF-VEGFR complex;an agent which inhibits PI3 Kinase activity; an agent that inhibitsVEGFR binding to p85, the catalytic subunit of PI3 kinase; an agentwhich inhibits AKT kinase activity.

[0016] In a preferred embodiment, VEGF is inhibited. VEGF can beinhibited by administering an agent which inhibits VEGF gene expression,protein production levels and/or activity. An agent which inhibits VEGFcan be one or more of: a VEGF binding protein, e.g., a soluble VEGFbinding protein, e.g., the ectodomain of a VEGF-receptor; an antibodythat specifically binds to the VEGF protein, e.g., an antibody thatdisrupts VEGF's ability to bind to its natural cellular target, e.g.,disrupts VEGF's ability to bind to a VEGF receptor, e.g., Flt1 (VEGFR1),Flk1/KDR (VEGFR2), NP-1, tie, tie-2, or Flt4 (VEGFR3); an antibody thatdisrupts the ability of a VEGF receptor to bind to VEGF; an antibody orsmall molecule which disrupts a complex formed by VEGF and its receptor;a mutated inactive VEGF or fragment which binds to a VEGF receptor butdoes not activate the receptor; a VEGF nucleic acid molecule which canbind to a cellular VEGF nucleic acid sequence, e.g., mRNA, and inhibitexpression of the protein, e.g., an antisense molecule or VEGF ribozyme;an agent which decreases VEGF gene expression, e.g., a small moleculewhich binds the promoter of VEGF. In another preferred embodiment, VEGFis inhibited by decreasing the level of expression of an endogenous VEGFgene, e.g., by decreasing transcription of the VEGF gene. In a preferredembodiment, transcription of the VEGF gene can be decreased by: alteringthe regulatory sequences of the endogenous VEGF gene, e.g., by theaddition of a negative regulatory sequence (such as a DNA-biding sitefor a transcriptional repressor), or by the removal of a positiveregulatory sequence (such as an enhancer or a DNA-binding site for atranscriptional activator). In another preferred embodiment, theantibody which binds VEGF is a monoclonal antibody, e.g., a humanizedchimeric or human monoclonal antibody.

[0017] In a preferred embodiment, VEGF interaction with its receptor isinhibited. An agent which inhibits a VEGF receptor, e.g., Flt1 (VEGFR1),Flk1/KDR (VEGFR2), neuropilin-1, tie, tie-2, or Flt4 (VEGFR3), can beone or more of: a VEGF receptor nucleic acid molecule which can bind toa cellular VEGF receptor nucleic acid sequence, e.g., mRNA, and inhibitexpression of the protein, e.g., an antisense molecule or VEGF receptorribozyme; an agent which decreases VEGF receptor gene expression, e.g.,a small molecule which binds the promoter of a VEGF receptor. In anotherpreferred embodiment, a VEGF receptor is inhibited by decreasing thelevel of expression of an endogenous VEGF receptor gene, e.g., bydecreasing transcription of an VEGF receptor gene. In a preferredembodiment, transcription of a VEGF receptor gene can be decreased by:altering the regulatory sequences of an endogenous VEGF receptor gene,e.g., by the addition of a negative regulatory sequence (such as aDNA-biding site for a transcriptional repressor), or by the removal of apositive regulatory sequence (such as an enhancer or a DNA binding sitefor a transcriptional activator); as well as agents described above.

[0018] In a preferred embodiment, PI3 kinase is inhibited. An agentwhich inhibits PI3-kinase activity can be one or more of: a smallmolecule which inhibits PI3-kinase activity, e.g., LY294002; a proteinor peptide that inhibits PI3 kinase activity, e.g., a PI3 kinase bindingprotein which binds to PI3-kinase but does not activate the enzyme, or adominant negative form of p85; an antibody that specifically binds tothe PI3-kinase protein, e.g., an antibody that disrupts PI3-kinase'scatalytic activity or an antibody that disrupts the ability of cellularreceptors to activate PI3-kinase; a PI3 kinase nucleic acid moleculewhich can bind to a cellular PI3 kinase nucleic acid sequence, e.g.,mRNA, and inhibit expression of the protein, e.g., an antisense moleculeor PI3-kinase ribozyme; an agent which decreases PI3-kinase geneexpression, e.g., a small molecule which binds the promoter ofPI3-kinase. In another preferred embodiment, PI3-kinase is inhibited bydecreasing the level of expression of an endogenous PI3-kinase gene,e.g., by decreasing transcription of the PI3-kinase gene. In a preferredembodiment, transcription of the PI3-kinase gene can be decreased by:altering the regulatory sequences of the endogenous PI3-kinase gene,e.g., by the addition of a negative regulatory sequence (such as aDNA-biding site for a transcriptional repressor), or by the removal of apositive regulatory sequence (such as an enhancer or a DNA-binding sitefor a transcriptional activator). In another preferred embodiment,PI3-kinase activity is inhibited by a specific small molecule inhibitor,e.g., wortmannin or LY294002.

[0019] In another preferred embodiment, AKT kinase is inhibited. Anagent which inhibits AKT kinase activity can be one or more of: aspecific small molecule which inhibits AKT activity; an AKT bindingprotein which binds to AKT but does not activate the enzyme; an antibodythat specifically binds to the AKT protein, e.g., an antibody thatdisrupts AKT's catalytic activity or an antibody that disrupts theability of the AKT PH domain to sense activating second messengers,e.g., phosphoinositides; a mutated inactive AKT or fragment which bindsto a AKT receptor but does not activate the receptor; an AKT nucleicacid molecule which can bind to a cellular AKT nucleic acid sequence,e.g., mRNA, and inhibit expression of the protein, e.g., an antisensemolecule or AKT ribozyme; an agent which decreases AKT gene expression,e.g., a small molecule which binds the promoter of AKT. In anotherpreferred embodiment, AKT is inhibited by decreasing the level ofexpression of an endogenous AKT gene, e.g., by decreasing transcriptionof the AKT gene. In a preferred embodiment, transcription of the AKTgene can be decreased by: altering the regulatory sequences of theendogenous AKT gene, e.g., by the addition of a negative regulatorysequence (such as a DNA-biding site for a transcriptional repressor), orby the removal of a positive regulatory sequence (such as an enhancer ora DNA-binding site for a transcriptional activator).

[0020] In one embodiment, increasing a PKC activity, e.g., a PKCα,PKCβ1, PKCβ2, or PKCγ activity, can inhibit PI3 kinase or Akt, thusdecreasing CTGF expression or activity. The agent which increases thelevel of PKC activity can be one or more of the following: a smallmolecule which stimulates PKC activity, e.g., PMA; a PKC polypeptide ora functional fragment or analog thereof; a nucleotide sequence encodinga PKC polypeptide or functional fragment or analog thereof; an agentwhich increases PKC nucleic acid expression; e.g., a small moleculewhich binds to the promoter region of PKC. In a preferred embodiment,PKC levels are increased by administering, e.g., introducing, anucleotide sequence encoding a PKC polypeptide or functional fragment oranalog thereof, into a particular cell, e.g., an endothelial cell, afibroblast, or a pericyte, in the subject. The nucleotide sequence canbe a genome sequence or a cDNA sequence. The nucleotide sequence caninclude: a PKC coding region; a promoter sequence, e.g., a promotersequence from a PKC gene or from another gene; an enhancer sequence;untranslated regulatory sequences, e.g., a 5′untranslated region (UTR),e.g., a 5′UTR from a PKC gene or from another gene, a 3′UTR, e.g., a3′UTR from a PKC gene or from another gene; a polyadenylation site; aninsulator sequence. In another preferred embodiment, the level of PKCprotein is increased by increasing the level of expression of anendogenous PKC gene, e.g., by increasing transcription of the PKC gene.In a preferred embodiment, transcription of the PKC gene is increasedby: altering the regulatory sequence of the endogenous PKC gene, e.g.,by the addition of a positive regulatory element (such as an enhancer ora DNA-binding site for a transcriptional activator); the deletion of anegative regulatory element (such as a DNA-binding site for atranscriptional repressor) and/or replacement of the endogenousregulatory sequence, or elements therein, with that of another gene,thereby allowing the coding region of the PKC gene to be transcribedmore efficiently.

[0021] In another preferred embodiment, Erk kinase is inhibited. Anagent which inhibits Erk kinase activity can be one or more of: aspecific small molecule which inhibits Erk activity; a Erk bindingprotein which binds to Erk but does not activate the enzyme; an antibodythat specifically binds to the Erk protein, e.g., an antibody thatdisrupts Erk's catalytic activity; a mutated inactive Erk or fragmentwhich binds to a Erk receptor but does not activate the receptor; a Erknucleic acid molecule which can bind to a cellular Erk nucleic acidsequence, e.g., mRNA, and inhibit expression of the protein, e.g., anantisense molecule or Erk ribozyme; an agent which decreases Erk geneexpression, e.g., a small molecule which binds the promoter of Erk. Inanother preferred embodiment, Erk is inhibited by decreasing the levelof expression of an endogenous Erk gene, e.g., by decreasingtranscription of the Erk gene. In a preferred embodiment, transcriptionof the Erk gene can be decreased by: altering the regulatory sequencesof the endogenous Erk gene, e.g., by the addition of a negativeregulatory sequence (such as a DNA-biding site for a transcriptionalrepressor), or by the removal of a positive regulatory sequence (such asan enhancer or a DNA-binding site for a transcriptional activator).

[0022] Another aspect of the invention features methods of reducingangiogenesis by decreasing CTGF activity or expression. CTGF activity orexpression is decreased by decreasing a VEGF signaling activity by anyof the methods described herein. In preferred embodiments, angiogenesisid reduced to, e.g., treat a disorder related to excessive angiogenesis,e.g., tumor growth, tumor metastasis, arthritis, retinal neovasculardisease, and retinal ischemia. The method includes administering anagent which decreases CTGF transcription or activity by inhibiting acomponent of the VEGF signaling pathway, e.g., by any of the agentsmentioned above.

[0023] Another aspect of the invention features methods of increasingfibrosis.

[0024] In a preferred embodiment, the invention features a method oftreating disorders related to insufficient fibrosis. The disorder can bethe result of an injury. The disorder can be due to a geneticdeficiency, a second physiological disorder or disease, or anenvironmental insult. In a preferred embodiment, the disorder is awound. In another preferred embodiment the disorder is a damaged organ,e.g., an organ undergoing regeneration. The method includesadministering an agent which increases the level of CTGF transcription.

[0025] An agent which increases the level of CTGF transcription can beone or more of: an agent which increases the level or activity of VEGF,e.g., a transition metal ion, e.g., manganese, cobalt, nickel, orcombinations thereof; an agent which activates the VEGF receptor; anagent which increases PI3 Kinase activity; an agent which increases AKTkinase activity.

[0026] In a preferred embodiment, VEGF is increased. An agent whichincreases the level of VEGF activity can be one or more of thefollowing; a small molecule, e.g., a transition metal ion; a peptide orprotein, e.g., a monoclonal antibody, which stabilizes or assists thebinding of VEGF to a VEGF receptor; a VEGF polypeptide or a functionalfragment or analog thereof; a nucleotide sequence encoding a VEGFpolypeptide or functional fragment or analog thereof; an agent whichincrease VEGF nucleic acid expression; e.g., a small molecule whichbinds to the promoter region of VEGF. In a preferred embodiment, VEGFlevels are increased by administering, e.g., introducing, a nucleotidesequence encoding a VEGF polypeptide or functional fragment or analogthereof, into a particular cell, e.g., an endothelial cell, afibroblast, or a pericyte, in the subject. The nucleotide sequence canbe a genome sequence or a cDNA sequence. The nucleotide sequence caninclude: a VEGF coding region; a promoter sequence, e.g., a promotersequence from a VEGF gene or from another gene; an enhancer sequence;untranslated regulatory sequences, e.g., a 5′untranslated region (UTR),e.g., a 5′UTR from a VEGF gene or from another gene, a 3′UTR, e.g., a3′UTR from a VEGF gene or from another gene; a polyadenylation site; aninsulator sequence. In another preferred embodiment, the level of VEGFprotein is increased by increasing the level of expression of anendogenous VEGF gene, e.g., by increasing transcription of the VEGFgene. In a preferred embodiment, transcription of the VEGF gene isincreased by: altering the regulatory sequence of the endogenous VEGFgene, e.g., by the addition of a positive regulatory element (such as anenhancer or a DNA-binding site for a transcriptional activator); thedeletion of a negative regulatory element (such as a DNA-binding sitefor a transcriptional repressor)and/or replacement of the endogenousregulatory sequence, or elements therein, with that of another gene,thereby allowing the coding region of the VEGF gene to be transcribedmore efficiently.

[0027] In a preferred embodiment, the VEGF receptor activity isincreased. An agent which increases the level of VEGF receptor activitycan be one or more of the following: an agent which activates a VEGFreceptor, e.g., a monoclonal antibody which activates the VEGF receptor,e.g., a monoclonal antibody which activates a VEGF receptor in theabsence of VEGF; a VEGF receptor ligand polypeptide (e.g., VEGF orplacenta growth factor (P1GF)), or a functional fragment or analogthereof; a nucleotide sequence encoding a VEGF receptor polypeptide orfunctional fragment or analog thereof; an agent which increase VEGFreceptor nucleic acid expression; e.g., a small molecule which binds tothe promoter region of VEGF receptor. In a preferred embodiment, VEGFreceptor levels are increased by administering, e.g., introducing, anucleotide sequence encoding a VEGF receptor polypeptide or functionalfragment or analog thereof, into a particular cell, e.g., an endothelialcell, a fibroblast, or a pericyte in the subject. The nucleotidesequence can be a genome sequence or a cDNA sequence. The nucleotidesequence can include: a VEGF receptor coding region; a promotersequence, e.g., a promoter sequence from a VEGF receptor gene or fromanother gene; an enhancer sequence; untranslated regulatory sequences,e.g., a 5′untranslated region (UTR), e.g., a 5′UTR from a VEGF receptorgene or from another gene, a 3′UTR, e.g., a 3′UTR from a VEGF receptorgene or from another gene; a polyadenylation site; an insulatorsequence. In another preferred embodiment, the level of VEGF receptorprotein is increased by increasing the level of expression of anendogenous VEGF receptor gene, e.g., by increasing transcription of theVEGF receptor gene. In a preferred embodiment, transcription of the VEGFreceptor gene is increased by: altering the regulatory sequence of theendogenous VEGF receptor gene, e.g., by the addition of a positiveregulatory element (such as an enhancer or a DNA-binding site for atranscriptional activator); the deletion of a negative regulatoryelement (such as a DNA binding site for a transcriptional repressor)and/or replacement of the endogenous regulatory sequence, or elementstherein, with that of another gene, thereby allowing the coding regionof the VEGF receptor gene to be transcribed more efficiently.

[0028] In a preferred embodiment, PI3-Kinase is increased. An agentwhich increases the level of PI3-kinase can be one or more of thefollowing: a small molecule which activates PI3kinase; a PI3kinasepolypeptide or a functional fragment or analog thereof; a nucleotidesequence encoding a PI3kinase polypeptide or functional fragment oranalog thereof; an agent which increase PI3-kinase nucleic acidexpression, e.g., a small molecule which binds to the promoter region ofPI3 kinase. In a preferred embodiment, PI3-kinase levels are increasedby administering, e.g., introducing, a nucleotide sequence encoding aPI3-kinase polypeptide or functional fragment or analog thereof, into aparticular cell, e.g., an endothelial cell, a fibroblast, or a pericyte,in the subject. The nucleotide sequence can be a genome sequence or acDNA sequence. The nucleotide sequence can include: a PI3-kinase codingregion; a promoter sequence, e.g., a promoter sequence from a PI3 kinasegene or from another gene; an enhancer sequence; untranslated regulatorysequences, e.g., a 5′untranslated region (UTR), e.g., a 5′UTR from aPI3kinase gene or from another gene, a 3′UTR, e.g., a 3′UTR from aPI3-kinase gene or from another gene; a polyadenylation site; aninsulator sequence. In another preferred embodiment, the level ofPI3kinase protein is increased by increasing the level of expression ofan endogenous PI3-kinase gene, e.g., by increasing transcription of thePI3-kinase gene. In a preferred embodiment, transcription of the PI3-kinase gene is increased by: altering the regulatory sequence of theendogenous PI3 kinase gene, e.g., by the addition of a positiveregulatory element (such as an enhancer or a DNA-binding site for atranscriptional activator); the deletion of a negative regulatoryelement (such as a DNA-binding site for a transcriptionalrepressor)and/or replacement of the endogenous regulatory sequence, orelements therein, with that of another gene, thereby allowing the codingregion of the PI3-kinase gene to be transcribed more efficiently.

[0029] In a preferred embodiment, AKT is increased. An agent whichincreases the level of AKT can be one or more of the following: a smallmolecule which activates AKT kinase activity, e.g., the phosphoinositidePIP₂; a polypeptide, e.g., insulin, which stimulates activation, e.g.,phosphorylation, of AKT; a AKT polypeptide or a functional fragment oranalog thereof; a nucleotide sequence encoding a AKT polypeptide orfunctional fragment or analog thereof; an agent which increase AKTnucleic acid expression; e.g., a small molecule which binds to thepromoter region of AKT. In a preferred embodiment, AKT levels areincreased by administering, e.g., introducing, a nucleotide sequenceencoding a AKT polypeptide or functional fragment or analog thereof,into a particular cell, e.g., an endothelial cell, a fibroblast, or apericyte, in the subject. The nucleotide sequence can be a genomesequence or a cDNA sequence. The nucleotide sequence can include: a AKTcoding region; a promoter sequence, e.g., a promoter sequence from a AKTgene or from another gene; an enhancer sequence; untranslated regulatorysequences, e.g., a 5′untranslated region (UTR), e.g., a 5′UTR from a AKTgene or from another gene, a 3′UTR, e.g., a 3′UTR from a AKT gene orfrom another gene; a polyadenylation site; an insulator sequence. Inanother preferred embodiment, the level of AKT protein is increased byincreasing the level of expression of an endogenous AKT gene, e.g., byincreasing transcription of the AKT gene. In a preferred embodiment,transcription of the AKT gene is increased by: altering the regulatorysequence of the endogenous AKT gene, e.g., by the addition of a positiveregulatory element (such as an enhancer or a DNA-binding site for atranscriptional activator); the deletion of a negative regulatoryelement (such as a DNA-binding site for a transcriptional repressor)and/or replacement of the endogenous regulatory sequence, or elementstherein, with that of another gene, thereby allowing the coding regionof the AKT gene to be transcribed more efficiently.

[0030] In another preferred embodiment, Erk kinase is increased. Anagent which increases Erk kinase activity can be one or more of: a smallmolecule which activates Erk kinase activity; an Erk polypeptide or afunctional fragment or analog thereof; a nucleotide sequence encoding anErk polypeptide or functional fragment or analog thereof; an agent whichincreases Erk nucleic acid expression; e.g., a small molecule whichbinds to the promoter region of Erk. In a preferred embodiment, Erklevels are increased by administering, e.g., introducing, a nucleotidesequence encoding an Erk polypeptide or functional fragment or analogthereof, into a particular cell, e.g., an endothelial cell, afibroblast, or a pericyte, in the subject. The nucleotide sequence canbe a genome sequence or a cDNA sequence. The nucleotide sequence caninclude: an Erk coding region; a promoter sequence, e.g., a promotersequence from an Erk gene or from another gene; an enhancer sequence;untranslated regulatory sequences, e.g., a 5′untranslated region (UTR),e.g., a 5′UTR from an Erk gene or from another gene, a 3′UTR, e.g., a3′UTR from an Erk gene or from another gene; a polyadenylation site; aninsulator sequence. In another preferred embodiment, the level of Erkprotein is increased by increasing the level of expression of anendogenous Erk gene, e.g., by increasing transcription of the Erk gene.In a preferred embodiment, transcription of the Erk gene is increasedby: altering the regulatory sequence of the endogenous Erk gene, e.g.,by the addition of a positive regulatory element (such as an enhancer ora DNA-binding site for a transcriptional activator); the deletion of anegative regulatory element (such as a DNA-binding site for atranscriptional repressor) and/or replacement of the endogenousregulatory sequence, or elements therein, with that of another gene,thereby allowing the coding region of the Erk gene to be transcribedmore efficiently.

[0031] Another aspect of the invention features a method of screeningfor agents which increase or decrease fibrosis and/or angiogenesis tothereby treat disorders associated with increased or decreased fibrosis,e.g., the disorders mentioned above. The method includes: providing acell, tissue, or subject, e.g., an experimental animal, e.g., an animalmodel for a fibrosis related disorder; contacting the cell, tissue orsubject with a test agent; and determining whether the test agentinhibits a component of the VEGF signaling pathway.

[0032] In preferred embodiments, the screening can include screeningfor: an agent that inhibits VEGF activity; an agent that inhibits VEGFRsignaling, e.g., an agent that inhibits the interaction between VEGF anda VEGFR; an agent that inhibits PI3 kinase activity; an agent thatinhibits a VEGFR interaction with p85 subunit of PI3-kinase; an agentthat inhibits AKT activity; and/or an agent that inhibits ERK activity.

[0033] In some embodiments, the method can include one or more of thefollowing steps: applying VEGF to cells in culture, e.g., BREC or BREP;applying a candidate drug or a combinatorial library of drugs; assayingfor levels of CTGF. CTGF levels can be assayed various methods commonlypracticed in the art. In one embodiment, CTGF levels are assayed byNorthern analysis for CTGF mRNA expression. In another embodiment CTGFlevels are assayed by detect CTGF protein, e.g., with an antibody, e.g.,using an ELISA assay or a Western blot assay. In another embodiment,CTGF transcription is monitored by assaying for a reporter protein,e.g., lacZ, chloramphenicol acetyltransferase (CAT), green fluorescentprotein or variants thereof, and other fluorescent proteins and variantsthereof, where the gene encoding the reporter protein is fused to theCTGF promoter and the ensemble is transfected into the cells. Themethods can further include administering an agent identified by thescreening methods described herein to an animal, e.g., an animal modelfor a fibrotic disorder.

DETAILED DESCRIPTION

[0034] The inventors have discovered that VEGF can modulate the activityand/or expression of CTGF in a time- and concentration-dependent mannerin cells and/or tissues with VEGF receptors, e.g., in endothelial cells,e.g., microvascular endothelial cells such as human retinal endothelialcells or bovine retinal endothelial cells (BREC), and in contractilecells, e.g., in capillary pericytes, e.g., in human or bovine retinalpericytes (BRPC). VEGF-induced modulation of CTGF can occur via the PI3kinase pathway, e.g., via the KDR receptor-PI3 kinase-Erk pathway (e.g.,in BREC), or the Flt1-PI3 kinase-Akt pathway (e.g., in BRPC). Modulationof CTGF via the VEGF pathway can be used to modulate fibrosis and/orangiogenesis, e.g., in the treatment of fibrosis-related disordersdescribed herein.

[0035] VEGF receptor (VEGFR), e.g., Flt1 (VEGFR1), KDR/Flk1 (VEGFR2),Flt4 (VEGFR3), NP1, tie, or tie-2, can mediate increases in CTGF mRNAexpression. The ability of Flt1 to induce increases in CTGF mRNA levelsis demonstrated in the pericytes that have predominantly Flt1 receptors.In addition, placenta growth factor (P1GF), a Flt1 receptor-specificligand, was able to induce CTGF mRNA levels in BRPC but not in BREC,indicating that VEGF can induce CTGF mRNA by activating through Flt1 inpericytes, e.g., BRPC. The KDR/Flk1 receptors in the endothelial cellscan also induce CTGF gene expression since KDR/Flk1 receptors are thepredominant VEGF receptors in endothelial cells.

[0036] The VEGF dose-response curves for CTGF in both BRPC and BREC aresimilar and suggest that VEGF binds to high affinity receptors,consistent with the known Kd values of Flt1 and KDR/Flk1 at 10-100 pM.While not wanting to be bound by theory, VEGF-induced CTGF mRNA is mostlikely due to an induction of transcription rather than altering thehalf-life of CTGF mRNA since the addition of VEGF failed to change thedegradation rates of CTGF mRNA. The time course of the action of VEGF onCTGF (which required 6-9 h) suggests this is potentially a chronicaction of VEGF. In addition, the time needed to achieve maximum effectis also consistent with the calculated mRNA half-life of CTGF mRNA of2-4 h. From a biological perspective, the effects of VEGF on CTGF mRNAcould potentially have important physiological impact for severalreasons. First, the increase in CTGF mRNA results in increased proteinlevels. Second, the VEGF concentration that was minimally active (0.25ng/ml) can easily bind and activate a significant percentage of theVEGFR-1, -2 receptors. Third, this low level of VEGF may exist even innon-pathological states, suggesting that low levels of VEGF may havephysiological actions on maintaining extracellular matrix production viathe induction of CTGF. At 2.5-25 ng/ml VEGF which are encountered inhypoxic and angiogenic states (Aiello et al. (1994) N. Engl. J. Med.331, 1480-1487), the induction of CTGF expression by VEGF couldpotentially induce the fibrosis that frequently accompaniesneovascularization. This possibility is supported further by thedemonstration that the protein levels of CTGF expression were increased10 h after the addition of VEGF that was consistent with the maximumincrease in the mRNA levels at 6-9 h. In addition, the potency of VEGFon CTGF expression appeared to be similar to TGF-betal, suggesting thatboth of them could induce fibrosis associated with neovascularization.

[0037] The activation of the endogenous tyrosine kinases of KDR/Flk1scan stimulate multiple signaling pathways, including Ras-Erk,PI3-kinase-Akt, and phospholipase CK-PKC cascades. The results in BRECdescribed herein indicate that VEGF can increase the tyrosinephosphorylation of KDR/Flk1 and its interaction with p85 subunit ofPI3-kinase. In addition, VEGF also activates the Erk1/2 pathway in BREC.In contrast, VEGF was unable to activate Erk1/2 but stimulated theactivation of PI3-kinase and phosphorylation of Akt in BRPC. Theseresults indicate that the signaling pathways for Flt1 in vascular cellsare different from those for KDR/Flk1. The lack of effect on Erk1/2activation also supports the hypothesis that Flt1, unlike KDR/Flk1, isnot involved in mitogenic actions.

[0038] The results described herein provide strong evidence that VEGF isinducing CTGF gene expression in both endothelial cells and pericytesvia VEGFR1 or -R2 by the activation of PI3-kinase and Akt. This evidenceincludes the ability of wortmannin, a PI3-kinase inhibitor, to inhibitthe effects of VEGFs in both cell types. Adenovirus containing dominantnegative mutants of p85 subunit of PI3-kinase or Akt inhibited theaction of VEGFs, whereas overexpression of dominant negative mutants ofRas and Erk1 by adenovirus vectors did not inhibit CTGF mRNA expression.Conversely, the overexpression of constitutive active Akt increased CTGFmRNA expression by 2.5-fold. The overexpression of either the wild typeor dominant negative of PKC isoform did not alter the effects of VEGF onCTGF mRNA levels.

[0039] In summary, the results described herein show that VEGF caninduce the expression of CTGF via VEGFR, e.g., Flt1, KDR/Flk1, Flt4,NP-1, tie, or tie-2, by the selectively activated PI3-kinase-Akt pathwaybut mostly independent of the Ras-Erk pathway. In addition, the spectrumof signaling pathways can be different among different VEGFRs, possiblyreflecting their physiological roles. These results support theconclusion that VEGF, through its effects on CTGF expression, hasphysiological roles such as the maintenance of capillary strength andwound healing via the extracellular matrix production. In diseasestates, VEGF-induced CTGF may cause the proliferation of fibrocellularcomponents in retinal neovascular diseases such as proliferativediabetic retinopathy and age-related macular degeneration.

[0040] Modulation of CTGF through the VEGF pathway, e.g., modulation ofcomponents of the VEGF pathway, e.g., Flt1, KDR, Flt4, neuropilin-1, PI3kinase, Akt, or Erk, can be used to modulate fibrosis and/orangiogenesis, e.g., in the treatment of fibrosis related disorders. CTGFhas been associated with a number of fibrosis-related disorders, e.g.,scleroderma (e.g., morphea, generalized morphea, linear scleroderma);keloids; kidney fibrosis, e.g., glomerular sclerosis or renaltubulointerstitial fibrosis; pulmonary fibrosis; cardiac fibrosis;chemotherapy/radiation induced lung fibrosis; pancreatitis; renaldisease; atherosclerotic plaques; inflammatory bowel disease; Crohn'sdisease; arthritic joints; cancer, e.g., invasive breast carcinoma,dermatofibromas, angiolipoma, and angioleiomyoma; hypertrophic scar;nodular fasciitis, eosinophilic fasciitis, dupuytren's contracture (J.Invest. Dermatol. 1996 106:729-733; J Biol Chem. 2001 276:10594-601; IntJ Biochem Cell Biol. 1998 30:909-22; J Eur Acad Dermatol Venereol. 199811:1-8; Int J Biochem Cell Biol. 1998 Aug; 30(8):909-22; Ann Surg. 1999Jul;230(1):63-71; J Cell Physiol. 1999 Oct;181(1):153-9; Curr OpinNephrol Hypertens. 1999 Sep;8(5):543-8; J Am Soc Nephrol. 2001Mar;12(3):472-84; J Rheumatol. 2000 Jan;27(1): 149-54; J Mol CellCardiol. 2000 Oct;32(10): 1805-19). Other fibrosis related diseases inwhich CTGF may be involved include macular degeneration, e.g.,age-related macular degeneration; acute ocular neovascularization ordiabetic retinopathy; general fibrosis syndrome, characterized byreplacement of normal muscle tissue by fibrous tissue in varyingdegrees; retroperitoneal fibrosis; liver fibrosis; acute fibrosis, e.g.,in response to various forms of trauma including accidental injuries,infections, surgery, burns, radiation or chemotherapy treatments.

[0041] Generation of Fragments

[0042] Fragments of components of the VEGF signaling pathway, e.g.,VEGF, VEGFRs, PI3 kinase, AKT, can be used to increase VEGF signaling,thereby increasing CTGF activity, thereby increasing fibrosis orangiogenesis. For example, various fragments of VEGF are known anddescribed, for example, in U.S. Pat. No.: 5,935,820.

[0043] Fragments of a protein can be produced in several ways, e.g.,recombinantly, by proteolytic digestion, or by chemical synthesis.Internal or terminal fragments of a polypeptide can be generated byremoving one or more nucleotides from one end (for a terminal fragment)or both ends (for an internal fragment) of a nucleic acid which encodesthe polypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end-nibbling” endonucleases can thus generateDNA's which encode an array of fragments. DNA's which encode fragmentsof a protein can also be generated by random shearing, restrictiondigestion or a combination of the above-discussed methods.

[0044] Fragments can also be chemically synthesized using techniquesknown in the art such as conventional Merrifield solid phase f-Moc ort-Boc chemistry. For example, peptides of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

[0045] Generation of Analogs: Production of Altered DNA and PeptideSequences by Random Methods

[0046] Amino acid sequence variants of a protein, e.g., a VEGF signalingpathway component described herein, can be prepared by randommutagenesis of DNA which encodes a protein or a particular domain orregion of a protein. Useful methods include PCR mutagenesis andsaturation mutagenesis. A library of random amino acid sequence variantscan also be generated by the synthesis of a set of degenerateoligonucleotide sequences. (Methods for screening proteins in a libraryof variants, e.g., screening for CTGF modulating activity or VEGFsignaling agonist or antagonist activity, are elsewhere herein.)

[0047] PCR Mutagenesis

[0048] In PCR mutagenesis, reduced Taq polymerase fidelity is used tointroduce random mutations into a cloned fragment of DNA (Leung et al.,1989, Technique 1:11-15). This is a very powerful and relatively rapidmethod of introducing random mutations. The DNA region to be mutagenizedis amplified using the polymerase chain reaction (PCR) under conditionsthat reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g.,by using a dGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction.The pool of amplified DNA fragments are inserted into appropriatecloning vectors to provide random mutant libraries.

[0049] Saturation Mutagenesis

[0050] Saturation mutagenesis allows for the rapid introduction of alarge number of single base substitutions into cloned DNA fragments(Mayers et al., 1985, Science 229:242). This technique includesgeneration of mutations, e.g., by chemical treatment or irradiation ofsingle-stranded DNA in vitro, and synthesis of a complimentary DNAstrand. The mutation frequency can be modulated by modulating theseverity of the treatment, and essentially all possible basesubstitutions can be obtained. Because this procedure does not involve agenetic selection for mutant fragments both neutral substitutions, aswell as those that alter function, are obtained. The distribution ofpoint mutations is not biased toward conserved sequence elements.

[0051] Degenerate Oligonucleotides

[0052] A library of homologs can also be generated from a set ofdegenerate oligonucleotide sequences. Chemical synthesis of a degeneratesequences can be carried out in an automatic DNA synthesizer, and thesynthetic genes then ligated into an appropriate expression vector. Thesynthesis of degenerate oligonucleotides is known in the art (see forexample, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981)Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. A GWalton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

[0053] Generation of Analogs: Production of Altered DNA and PeptideSequences by Directed Mutagenesis

[0054] Non-random or directed, mutagenesis techniques can be used toprovide specific sequences or mutations in specific regions. Thesetechniques can be used to create variants which include, e.g.,deletions, insertions, or substitutions, of residues of the known aminoacid sequence of a protein. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

[0055] Alanine Scanning Mutagenesis

[0056] Alanine scanning mutagenesis is a useful method foridentification of certain residues or regions of the desired proteinthat are preferred locations or domains for mutagenesis, Cunningham andWells (Science 244:1081-1085, 1989). In alanine scanning, a residue orgroup of target residues are identified (e.g., charged residues such asArg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine). Replacementof an amino acid can affect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Those domainsdemonstrating functional sensitivity to the substitutions are thenrefined by introducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted at the target codon or region and the expressed desiredprotein subunit variants are screened for the optimal combination ofdesired activity.

[0057] Oligonucleotide-Mediated Mutagenesis

[0058] Oligonucleotide-mediated mutagenesis is a useful method forpreparing substitution, deletion, and insertion variants of DNA, see,e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA isaltered by hybridizing an oligonucleotide encoding a mutation to a DNAtemplate, where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thedesired protein. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the desired protein DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. (1978) USA, 75: 5765).

[0059] Cassette Mutagenesis

[0060] Another method for preparing variants, cassette mutagenesis, isbased on the technique described by Wells et al. (Gene, 34:315[1985]).The starting material is a plasmid (or other vector) which includes theprotein subunit DNA to be mutated. The codon(s) in the protein subunitDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the desired protein subunit DNA. Afterthe restriction sites have been introduced into the plasmid, the plasmidis cut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are comparable with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated desired protein subunit DNAsequence.

[0061] Combinatorial Mutagenesis

[0062] Combinatorial mutagenesis can also be used to generate mutants.For example, the amino acid sequences for a group of homologs or otherrelated proteins are aligned, preferably to promote the highest homologypossible. All of the amino acids which appear at a given position of thealigned sequences can be selected to create a degenerate set ofcombinatorial sequences. The variegated library of variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential sequences are expressible asindividual peptides, or alternatively, as a set of larger fusionproteins containing the set of degenerate sequences.

[0063] Primary High-Through-Put Methods for Screening Libraries ofPeptide Fragments or Homologs

[0064] Various techniques are known in the art for screening generatedmutant gene products. Techniques for screening large gene librariesoften include cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors, and expressing the genes under conditions in which detection ofa desired activity, assembly into a trimeric molecules, binding tonatural ligands, e.g., a receptor or substrates, facilitates relativelyeasy isolation of the vector encoding the gene whose product wasdetected. Each of the techniques described below is amenable to highthrough-put analysis for screening large numbers of sequences created,e.g., by random mutagenesis techniques.

[0065] Two Hybrid Systems

[0066] Two hybrid (interaction trap) assays can be used to identify aprotein that interacts with a component of the VEGF signaling pathway,e.g., VEGF, VEGFR (e.g., flt1, flt4, KDR, neuropilin), PI3 kinase (e.g.,p85), AKT, ERK. These may include agonists, superagonists, andantagonists of the components. (The subject protein and a protein itinteracts with are used as the bait protein and fish proteins.). Theseassays rely on detecting the reconstitution of a functionaltranscriptional activator mediated by protein-protein interactions witha bait protein. In particular, these assays make use of chimeric geneswhich express hybrid proteins. The first hybrid comprises a DNA-bindingdomain fused to the bait protein. e.g., a VEGF or VEGFR molecule or afragment thereof. The second hybrid protein contains a transcriptionalactivation domain fused to a “fish” protein, e.g. an expression library.If the fish and bait proteins are able to interact, they bring intoclose proximity the DNA-binding and transcriptional activator domains.This proximity is sufficient to cause transcription of a reporter genewhich is operably linked to a transcriptional regulatory site which isrecognized by the DNA binding domain, and expression of the marker genecan be detected and used to score for the interaction of the baitprotein with another protein.

[0067] Display Libraries

[0068] In one approach to screening assays, the candidate peptides aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate receptorprotein via the displayed product is detected in a “panning assay”. Forexample, the gene library can be cloned into the gene for a surfacemembrane protein of a bacterial cell, and the resulting fusion proteindetected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991)Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).In a similar fashion, a detectably labeled ligand can be used to scorefor potentially functional peptide homologs. Fluorescently labeledligands, e.g., receptors, can be used to detect homolog which retainligand-binding activity. The use of fluorescently labeled ligands,allows cells to be visually inspected and separated under a fluorescencemicroscope, or, where the morphology of the cell permits, to beseparated by a fluorescence-activated cell sorter.

[0069] A gene library can be expressed as a fusion protein on thesurface of a viral particle. For instance, in the filamentous phagesystem, foreign peptide sequences can be expressed on the surface ofinfectious phage, thereby conferring two significant benefits. First,since these phage can be applied to affinity matrices at concentrationswell over 10¹³ phage per milliliter, a large number of phage can bescreened at one time. Second, since each infectious phage displays agene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd., and fl are most often used in phage display libraries.Either of the phage gIII or gVIII coat proteins can be used to generatefusion proteins without disrupting the ultimate packaging of the viralparticle. Foreign epitopes can be expressed at the NH₂-terminal end ofpIII and phage bearing such epitopes recovered from a large excess ofphage lacking this epitope (Ladner et al. PCT publication WO 90/02909;Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J.Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734;Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

[0070] A common approach uses the maltose receptor of E. coli (the outermembrane protein, LamB) as a peptide fusion partner (Charbit et al.(1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted intoplasmids encoding the LamB gene to produce peptides fused into one ofthe extracellular loops of the protein. These peptides are available forbinding to ligands, e.g., to antibodies, and can elicit an immuneresponse when the cells are administered to animals. Other cell surfaceproteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392),PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al.(1991) Bio/Tech 9, 1369-1372), as well as large bacterial surfacestructures have served as vehicles for peptide display. Peptides can befused to pilin, a protein which polymerizes to form the pilus-a conduitfor interbacterial exchange of genetic information (Thiry et al. (1989)Appl. Environ. Microbiol. 55, 984-993). Because of its role ininteracting with other cells, the pilus provides a useful support forthe presentation of peptides to the extracellular environment. Anotherlarge surface structure used for peptide display is the bacterial motiveorgan, the flagellum. Fusion of peptides to the subunit proteinflagellin offers a dense array of may peptides copies on the host cells(Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins ofother bacterial species have also served as peptide fusion partners.Examples include the Staphylococcus protein A and the outer membraneprotease IgA of Neisseria (Hansson et al. (1992)J. Bacteriol. 174,4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991-1999).

[0071] In the filamentous phage systems and the LamB system describedabove, the physical link between the peptide and its encoding DNA occursby the containment of the DNA within a particle (cell or phage) thatcarries the peptide on its surface. Capturing the peptide captures theparticle and the DNA within. An alternative scheme uses the DNA-bindingprotein LacI to form a link between peptide and DNA (Cull et al. (1992)PNAS USA 89:1865-1869). This system uses a plasmid containing the LacIgene with an oligonucleotide cloning site at its 3′-end. Under thecontrolled induction by arabinose, a LacI-peptide fusion protein isproduced. This fusion retains the natural ability of LacI to bind to ashort DNA sequence known as LacO operator (LacO). By installing twocopies of LacO on the expression plasmid, the LacI-peptide fusion bindstightly to the plasmid that encoded it. Because the plasmids in eachcell contain only a single oligonucleotide sequence and each cellexpresses only a single peptide sequence, the peptides becomespecifically and stably associated with the DNA sequence that directedits synthesis. The cells of the library are gently lysed and thepeptide-DNA complexes are exposed to a matrix of immobilized receptor torecover the complexes containing active peptides. The associated plasmidDNA is then reintroduced into cells for amplification and DNA sequencingto determine the identity of the peptide ligands. As a demonstration ofthe practical utility of the method, a large random library ofdodecapeptides was made and selected on a monoclonal antibody raisedagainst the opioid peptide dynorphin B. A cohort of peptides wasrecovered, all related by a consensus sequence corresponding to asix-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl.Acad. Sci. U.S.A. 89-1869)

[0072] This scheme, sometimes referred to as peptides-on-plasmids,differs in two important ways from the phage display methods. First, thepeptides are attached to the C-terminus of the fusion protein, resultingin the display of the library members as peptides having free carboxytermini. Both of the filamentous phage coat proteins, pIII and pVIII,are anchored to the phage through their C-termini, and the guestpeptides are placed into the outward-extending N-terminal domains. Insome designs, the phage-displayed peptides are presented right at theamino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl.Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set ofbiological biases affecting the population of peptides actually presentin the libraries. The LacI fusion molecules are confined to thecytoplasm of the host cells. The phage coat fusions are exposed brieflyto the cytoplasm during translation but are rapidly secreted through theinner membrane into the periplasmic compartment, remaining anchored inthe membrane by their C-terminal hydrophobic domains, with theN-termini, containing the peptides, protruding into the periplasm whileawaiting assembly into phage particles. The peptides in the LacI andphage libraries may differ significantly as a result of their exposureto different proteolytic activities. The phage coat proteins requiretransport across the inner membrane and signal peptidase processing as aprelude to incorporation into phage. Certain peptides exert adeleterious effect on these processes and are underrepresented in thelibraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). Theseparticular biases are not a factor in the LacI display system.

[0073] The number of small peptides available in recombinant randomlibraries is enormous. Libraries of 10⁷-10⁹ independent clones areroutinely prepared. Libraries as large as 10¹¹ recombinants have beencreated, but this size approaches the practical limit for clonelibraries. This limitation in library size occurs at the step oftransforming the DNA containing randomized segments into the hostbacterial cells. To circumvent this limitation, an in vitro system basedon the display of nascent peptides in polysome complexes has recentlybeen developed. This display library method has the potential ofproducing libraries 3-6 orders of magnitude larger than the currentlyavailable phage/phagemid or plasmid libraries. Furthermore, theconstruction of the libraries, expression of the peptides, andscreening, is done in an entirely cell-free format.

[0074] In one application of this method (Gallop et al. (1994) J. Med.Chem. 37(9):1233-1251), a molecular DNA library encoding 10¹²decapeptides was constructed and the library expressed in an E. coli S30in vitro coupled transcription/translation system. Conditions werechosen to stall the ribosomes on the mRNA, causing the accumulation of asubstantial proportion of the RNA in polysomes and yielding complexescontaining nascent peptides still linked to their encoding RNA. Thepolysomes are sufficiently robust to be affinity purified on immobilizedreceptors in much the same way as the more conventional recombinantpeptide display libraries are screened. RNA from the bound complexes isrecovered, converted to cDNA, and amplified by PCR to produce a templatefor the next round of synthesis and screening. The polysome displaymethod can be coupled to the phage display system. Following severalrounds of screening, cDNA from the enriched pool of polysomes was clonedinto a phagemid vector. This vector serves as both a peptide expressionvector, displaying peptides fused to the coat proteins, and as a DNAsequencing vector for peptide identification. By expressing thepolysome-derived peptides on phage, one can either continue the affinityselection procedure in this format or assay the peptides on individualclones for binding activity in a phage ELISA, or for binding specificityin a completion phage ELISA (Barret, et al. (1992) Anal. Biochem204,357-364). To identify the sequences of the active peptides onesequences the DNA produced by the phagemid host.

[0075] Secondary Screens

[0076] The high through-put assays described above can be followed bysecondary screens in order to identify further biological activitieswhich will, e.g., allow one skilled in the art to differentiate agonistsfrom antagonists. The type of a secondary screen used will depend on thedesired activity that needs to be tested. For example, an assay can bedeveloped in which the ability to inhibit an interaction between aprotein of interest (e.g., VEGF or PI3kinase) and a ligand (e.g., VEGFRand AKT, respectively) can be used to identify antagonists from a groupof peptide fragments isolated though one of the primary screensdescribed above.

[0077] Therefore, methods for generating fragments and analogs andtesting them for activity are known in the art. Once the core sequenceof interest is identified, it is routine to perform for one skilled inthe art to obtain analogs and fragments.

[0078] Peptide Mimetics

[0079] The invention also provides for reduction of the protein bindingdomains of the subject polypeptides, e.g., VEGF or VEGFR, to generatemimetics, e.g. peptide or non-peptide agents. See, for example, “Peptideinhibitors of human papillomavirus protein binding to retinoblastomagene protein” European patent applications EP 0 412 762 and EP 0 031080.

[0080] Non-hydrolyzable peptide analogs of critical residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

[0081] Fusion Proteins

[0082] Polypeptides for modulating the level of a component of the VEGFsignaling pathway can be fused to another protein or portion thereof.For example, a VEGF protein or antagonist or fragment thereof, can beoperably linked to another polypeptide moiety to enhance solubility.Examples of a protein which can be fused with a protein or portionsthereof include a plasma protein or fragment thereof, which can improvethe circulating half life. For example, the fusion protein can be a VEGFfragment-immunoglobulin (Ig) fusion protein in which the VEGF sequenceis fused to a sequence derived from the immunoglobulin superfamily.Several soluble fusion protein constructs have been disclosed whereinthe extracellular domain of a cell surface glycoprotein is fused withthe constant F(c) region of an immunoglobulin. For example, Capon et al.(1989) Nature 337(9):525-531, provide guidance on generating a longerlasting CD4 analog by fusing CD4 to an immunoglobulin (IgG1). See also,Capon et al., U.S. Pat. Nos.: 5,116,964 and 5,428,130 (CD4-IgG fusionconstructs); Linsley et al., U.S. Pat. No. 5,434,131 (CTLA4-IgG1 andB7-IgG1 fusion constructs); Linsley et al. (1991) J. Exp. Med.174:561-569 (CTLA4-IgG1 fusion constructs); and Linsley et al. (1991) J.Exp. Med. 173:721-730 (CD28-IgG1 and B7-IgG1 fusion constructs). Suchfusion proteins have proven useful for modulating receptor-ligandinteractions and reducing inflammation in vivo. For example, fusionproteins in which an extracellular domain of cell surface tumor necrosisfactor receptor (TNFR) proteins has been fused to an immunoglobulinconstant (Fc) region have been used in vivo. See, for example, Morelandet al. (1997) N. Engl. J Med. 337(3):141-147; and, van der Poll et al.(1997) Blood 89(10):3727-3734).

[0083] Antibodies

[0084] The invention also includes antibodies specifically reactive witha component of the VEGF signaling pathway described herein.Anti-protein/anti-peptide antisera or monoclonal antibodies can be madeas described herein by using standard protocols (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold SpringHarbor Press: 1988)).

[0085] A component of the VEGF signaling pathway described herein, or aportion or fragment thereof, can be used as an immunogen to generateantibodies that bind the component using standard techniques forpolyclonal and monoclonal antibody preparation. The full-lengthcomponent protein can be used or, alternatively, antigenic peptidefragments of the component can be used as immunogens.

[0086] Typically, a peptide is used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, a recombinant VEGF peptide, or a chemically synthesized VEGFpeptide or anagonist. See, e.g., U.S. Pat. No. 5,460,959; and co-pendingU.S. applications U.S. Ser. No. 08/334,797; U.S. Ser. No. 08/231,439;U.S. Ser. No. 08/334,455; and U.S. Ser. No. 08/928,881 which are herebyexpressly incorporated by reference in their entirety. The nucleotideand amino acid sequences of components of the VEGF signaling pathwaydescribed herein are known. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic VEGF preparation induces a polyclonal anti-VEGF antibodyresponse.

[0087] Antibodies to a component of the VEGF signaling pathway, orfragments thereof, can be used to inhibit the levels of such acomponent, thereby decreasing CTGF activity. Examples of antibodyfragments include F(v), Fab, Fab′ and F(ab′)₂ fragments which can begenerated by treating the antibody with an enzyme such as pepsin. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular protein with whichit immunoreacts.

[0088] Additionally, antibodies produced by genetic engineering methods,such as chimeric and humanized monoclonal antibodies, comprising bothhuman and non-human portions, which can be made using standardrecombinant DNA techniques, can be used. Such chimeric and humanizedmonoclonal antibodies can be produced by genetic engineering usingstandard DNA techniques known in the art, for example using methodsdescribed in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al.,Science 240:1041-1043, 1988; Liu et al., PNAS 84:3439-3443, 1987; Liu etal., J. Immunol. 139:3521-3526, 1987; Sun et al. PNAS 84:214-218, 1987;Nishimura et al., Canc. Res. 47:999-1005, 1987; Wood et al., Nature314:446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559,1988); Morrison, S. L., Science 229:1202-1207, 1985; Oi et al.,BioTechniques 4:214, 1986; Winter U.S. Pat. No. 5,225,539; Jones et al.,Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534, 1988; andBeidler et al., J. Immunol. 141:4053-4060, 1988.

[0089] In addition, a human monoclonal antibody directed against acomponent of the VEGF signaling pathway described herein can be madeusing standard techniques. For example, human monoclonal antibodies canbe generated in transgenic mice or in immune deficient mice engraftedwith antibody-producing human cells. Methods of generating such mice aredescribe, for example, in Wood et al. PCT publication WO 91/00906,Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCTpublication WO 92/03918; Kay et al. PCT publication WO 92/03917; Kay etal. PCT publication WO 93/12227; Kay et al. PCT publication 94/25585;Rajewsky et al. Pct publication WO 94/04667; Ditullio et al. PCTpublication WO 95/17085; Lonberg, N. et al. (1994) Nature 368:856-859;Green, L. L. et al. (1994) Nature Genet. 7:13-21; Morrison, S. L. et al.(1994) Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. (1993)Year Immunol 7:33-40; Choi et al. (1993) Nature Genet. 4:117-123;Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al. (1991) Eur JImmunol 21:1323-1326); Duchosal et al. PCT publication WO 93/05796; U.S.Pat. No. 5,411,749; McCune et al. (1988) Science 241:1632-1639),Kamel-Reid et al. (1988) Science 242:1706; Spanopoulou (1994) Genes &Development 8:1030-1042; Shinkai et al. (1992) Cell 68:855-868). A humanantibody-transgenic mouse or an immune deficient mouse engrafted withhuman antibody-producing cells or tissue can be immunized with acomponent of the VEGF signaling pathway described herein or an antigenicpeptide thereof and splenocytes from these immunized mice can then beused to create hybridomas. Methods of hybridoma production are wellknown.

[0090] Human monoclonal antibodies against components of the VEGFsignaling pathway described herein can also be prepared by constructinga combinatorial immunoglobulin library, such as a Fab phage displaylibrary or a scFv phage display library, using immunoglobulin lightchain and heavy chain cDNAs prepared from mRNA derived from lymphocytesof a subject. See, e.g., McCafferty et al. PCT publication WO 92/01047;Marks et al. (1991) J. Mol. Biol. 222:581-597; and Griffths et al.(1993) EMBO J 12:725-734. In addition, a combinatorial library ofantibody variable regions can be generated by mutating a known humanantibody. For example, a variable region of a human antibody known tobind VEGF, can be mutated, by for example using randomly alteredmutagenized oligonucleotides, to generate a library of mutated variableregions which can then be screened to bind to VEGF. Methods of inducingrandom mutagenesis within the CDR regions of immunoglobin heavy and/orlight chains, methods of crossing randomized heavy and light chains toform pairings and screening methods can be found in, for example, Barbaset al. PCT publication WO 96/07754; Barbas et al. (1992) Proc. Nat'lAcad. Sci. USA 89:4457-4461.

[0091] The immunoglobulin library can be expressed by a population ofdisplay packages, preferably derived from filamentous phage, to form anantibody display library. Examples of methods and reagents particularlyamenable for use in generating antibody display library can be found in,for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTpublication WO 92/18619; Dower et al. PCT publication WO 91/17271;Winter et al. PCT publication WO 92/20791; Markland et al. PCTpublication WO 92/15679; Breitling et al. PCT publication WO 93/01288;McCafferty et al. PCT publication WO 92/01047; Garrard et al. PCTpublication WO 92/09690; Ladner et al. PCT publication WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) HumAntibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffths et al. (1993) supra; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624 -628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982. Once displayed on the surface ofa display package (e.g., filamentous phage), the antibody library isscreened to identify and isolate packages that express an antibody thatbinds a component of the VEGF signaling pathway described herein. In apreferred embodiment, the primary screening of the library involvespanning with an immobilized component of the VEGF signaling pathwaydescribed herein and display packages expressing antibodies that bindimmobilized a component of the VEGF signaling pathway described hereinare selected.

[0092] Antisense Nucleic Acid Sequences

[0093] Nucleic acid molecules which are antisense to a nucleotideencoding a component of the VEGF signaling pathway described herein,e.g., VEGF, VEGFR (e.g., flt1, flt4, KDR, neuropilin), PI3Kinase, AKT,can be used as an agent which inhibits expression of the component. An“antisense” nucleic acid includes a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding the component, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can form hydrogen bonds with a sense nucleic acid. The antisensenucleic acid can be complementary to an entire coding strand, or to onlya portion thereof. For example, an antisense nucleic acid molecule whichantisense to the “coding region” of the coding strand of a nucleotidesequence encoding the component can be used.

[0094] The coding strand sequences encoding components of the VEGFsignaling pathway described herein are known. Given the coding strandsequences encoding these components, antisense nucleic acids can bedesigned according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to the entirecoding region of mRNA, but more preferably is an oligonucleotide whichis antisense to only a portion of the coding or noncoding region ofmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of the mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acidcan be constructed using chemical synthesis and enzymatic ligationreactions using procedures known in the art. For example, an antisensenucleic acid (e.g., an antisense oligonucleotide) can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Examplesof modified nucleotides which can be used to generate the antisensenucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest.

[0095] Administration

[0096] An agent which modulates the level of expression of a componentof the VEGF signaling pathway described herein can be administered to asubject by standard methods. For example, the agent can be administeredby any of a number of different routes including intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal(topical), and transmucosal. In one embodiment, the VEGF signalingpathway modulating agent can be administered topically.

[0097] The agent which modulates protein levels, e.g., nucleic acidmolecules, polypeptides, fragments or analogs, modulators, andantibodies (also referred to herein as “active compounds”) can beincorporated into pharmaceutical compositions suitable foradministration to a subject, e.g., a human. Such compositions typicallyinclude the nucleic acid molecule, polypeptide, modulator, or antibodyand a pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances are known. Except insofaras any conventional media or agent is incompatible with the activecompound, such media can be used in the compositions of the invention.Supplementary active compounds can also be incorporated into thecompositions.

[0098] A pharmaceutical composition can be formulated to be compatiblewith its intended route of administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0099] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixtures thereofThe proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

[0100] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a VEGF polypeptide or anti-VEGF antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0101] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0102] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0103] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0104] The nucleic acid molecules described herein can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al., PNAS 91:3054-3057, 1994). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can include a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0105] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0106] The agent which modulates the activity of a component of the VEGFsignaling pathway described herein can be administered by locallyadministration, e.g., topical administration. The agent can be appliedonce or it can be administered continuously, e.g., the agent isadministered with sufficient frequency such that the affect on the VEGFprotein level is maintained for a selected period, e.g., 5, 10, 20, 30,50, 90, 180, 365 days or more. The administration of an agent whichmodulates, e.g., increases or inhibits, the level of a component of theVEGF signaling pathway described herein, e.g., a VEGF polypeptide or ananti-VEGF antibody, can also be repeated.

[0107] Transition Metals

[0108] Transition metal ions have been shown to enhance expression ofthe VEGF gene thereby increasing VEGF protein levels. See U.S. Pat. No.:5,480,975. Thus, in one aspect, a transition metal ion can be used toincrease CTGF, thereby increasing fibrosis or angiogenesis, byincreasing expression of VEGF.

[0109] The transition metals are the group consisting of the fourth,fifth and sixth levels of the periodic table and which fill the dorbital. They include such elements as Ni, Co, Mn, Zn, V, Cr, Fe, Cu,Mo, etc. The preferred candidate metal ions for use are manganese,cobalt and nickel.

[0110] Selection of other appropriate metal ions involves determiningwhether, and at what level, the ion will induce VEGF expression.Particularly for systemic applications, selection also involves a reviewof toxicity. Suitable techniques for those determinations are providedbelow in U.S. Pat. No.: 5,480,795. Preferred ions are those with asubstantial range between VEGF induction and toxicity.

[0111] The transition metal can be administered internally (systemic orlocal administration) in the form of a salt or free ion in abiologically compatible tablet or capsule, gel, or liquid, or it can beadministered externally in the form of a biologically compatible powder,salve, liquid, or transdermal patch. Any physiologically acceptableanion such as chloride, sulfate, etc., can be included in thecomposition.

[0112] Appropriate release rates and dosages can be determined in orderto affect the targeted tissue without substantial systemic effect. Forexample, these parameters can be determined as described in U.S. Pat.No.: 5,480,795.

[0113] Gene Therapy

[0114] The gene constructs of the invention can also be used as a partof a gene therapy protocol to deliver nucleic acids encoding either anagonistic or antagonistic form of a component of the VEGF signalingpathway described herein. The invention features expression vectors forin vivo transfection and expression of a component of the VEGF signalingpathway described herein in particular cell types so as to reconstitutethe function of, or alternatively, antagonize the function of thecomponent in a cell in which that polypeptide is misexpressed.Expression constructs of such components may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the component gene to cells in vivo.Approaches include insertion of the subject gene in viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associated virus,and herpes simplex virus-1, or recombinant bacterial or eukaryoticplasmids. Viral vectors transfect cells directly; plasmid DNA can bedelivered with the help of, for example, cationic liposomes (lipofectin)or derivatized (e.g. antibody conjugated), polylysine conjugates,gramacidin S, artificial viral envelopes or other such intracellularcarriers, as well as direct injection of the gene construct or CaPO4precipitation carried out in vivo.

[0115] A preferred approach for in vivo introduction of nucleic acidinto a cell is by use of a viral vector containing nucleic acid, e.g. acDNA, encoding a component of the VEGF signaling pathway describedherein. Infection of cells with a viral vector has the advantage that alarge proportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

[0116] Retrovirus vectors and adeno-associated virus vectors can be usedas a recombinant gene delivery system for the transfer of exogenousgenes in vivo, particularly into humans. These vectors provide efficientdelivery of genes into cells, and the transferred nucleic acids arestably integrated into the chromosomal DNA of the host. The developmentof specialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). A replication defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include *Crip, *Cre, *2 and *Am. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0117] Another viral gene delivery system useful in the presentinvention utilizes adenovirus-derived vectors. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See, for example, Berkneret al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 d1324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells and can be used to infect a wide variety of celltypes, including epithelial cells (Rosenfeld et al. (1992) cited supra).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis in situwhere introduced DNA becomes integrated into the host genome (e.g.,retroviral DNA). Moreover, the carrying capacity of the adenoviralgenome for foreign DNA is large (up to 8 kilobases) relative to othergene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham(1986) J. Virol. 57:267).

[0118] Yet another viral vector system useful for delivery of thesubject gene is the adeno-associated virus (AAV). Adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. (1992) Curr. Topics in Micro. and Immunol. 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol, 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0119] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of acomponent of the VEGF signaling pathway described herein in the tissueof an animal. Most nonviral methods of gene transfer rely on normalmechanisms used by mammalian cells for the uptake and intracellulartransport of macromolecules. In preferred embodiments, non-viral genedelivery systems of the present invention rely on endocytic pathways forthe uptake of the subject gene by the targeted cell. Exemplary genedelivery systems of this type include liposomal derived systems,poly-lysine conjugates, and artificial viral envelopes. Otherembodiments include plasmid injection systems such as are described inMeuli et al. (2001) J Invest Dermatol. 116(1):131-135; Cohen et al.(2000) Gene Ther 7(22):1896-905; or Tam et al. (2000) Gene Ther7(21):1867-74.

[0120] In a representative embodiment, a gene encoding a component ofthe VEGF signaling pathway described herein can be entrapped inliposomes bearing positive charges on their surface (e.g., lipofectins)and (optionally) which are tagged with antibodies against cell surfaceantigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka20:547-551; PCT publication WO91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

[0121] In clinical settings, the gene delivery systems for thetherapeutic gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057).

[0122] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced in tact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0123] Cell Therapy

[0124] A component of the VEGF signaling pathway described herein canalso be increased in a subject by introducing into a cell, e.g., anendothelial call, fibroblast or a keratinocyte, a nucleotide sequencethat modulates the production of the component, e.g., a nucleotidesequence encoding a component polypeptide or functional fragment oranalog thereof, a promoter sequence, e.g., a promoter sequence from aVEGF gene or from another gene; an enhancer sequence, e.g., 5′untranslated region (UTR), e.g., a 5′ UTR from a VEGF gene or fromanother gene, a 3′ UTR, e.g., a 3′ UTR from a VEGF gene or from anothergene; a polyadenylation site; an insulator sequence; or another sequencethat modulates the expression of VEGF. The cell can then be introducedinto the subject.

[0125] Primary and secondary cells to be genetically engineered can beobtained form a variety of tissues and include cell types which can bemaintained propagated in culture. For example, primary and secondarycells include fibroblasts, keratinocytes, epithelial cells (e.g.,mammary epithelial cells, intestinal epithelial cells), endothelialcells, glial cells, neural cells, formed elements of the blood (e.g.,lymphocytes, bone marrow cells), muscle cells (myoblasts) and precursorsof these somatic cell types. Primary cells are preferably obtained fromthe individual to whom the genetically engineered primary or secondarycells are administered. However, primary cells may be obtained for adonor (other than the recipient).

[0126] The term “primary cell” includes cells present in a suspension ofcells isolated from a vertebrate tissue source (prior to their beingplated i.e., attached to a tissue culture substrate such as a dish orflask), cells present in an explant derived from tissue, both of theprevious types of cells plated for the first time, and cell suspensionsderived from these plated cells. The term “secondary cell” or “cellstrain” refers to cells at all subsequent steps in culturing. Secondarycells are cell strains which consist of secondary cells which have beenpassaged one or more times.

[0127] Primary or secondary cells of vertebrate, particularly mammalian,origin can be transfected with an exogenous nucleic acid sequence whichincludes a nucleic acid sequence encoding a signal peptide, and/or aheterologous nucleic acid sequence, e.g., encoding a component of theVEGF signaling pathway described herein or an agonist or antagonistthereof, and produce the encoded product stably and reproducibly invitro and in vivo, over extended periods of time. A heterologous aminoacid can also be a regulatory sequence, e.g., a promoter, which causesexpression, e.g., inducible expression or upregulation, of an endogenoussequence. An exogenous nucleic acid sequence can be introduced into aprimary or secondary cell by homologous recombination as described, forexample, in U.S. Pat. No.: 5,641,670, the contents of which areincorporated herein by reference. The transfected primary or secondarycells may also include DNA encoding a selectable marker which confers aselectable phenotype upon them, facilitating their identification andisolation.

[0128] Vertebrate tissue can be obtained by standard methods such apunch biopsy or other surgical methods of obtaining a tissue source ofthe primary cell type of interest. For example, punch biopsy is used toobtain skin as a source of fibroblasts or keratinocytes. A mixture ofprimary cells is obtained from the tissue, using known methods, such asenzymatic digestion or explanting. If enzymatic digestion is used,enzymes such as collagenase, hyaluronidase, dispase, pronase, trypsin,elastase and chymotrypsin can be used.

[0129] The resulting primary cell mixture can be transfected directly orit can be cultured first, removed from the culture plate and resuspendedbefore transfection is carried out. Primary cells or secondary cells arecombined with exogenous nucleic acid sequence to, e.g., stably integrateinto their genomes, and treated in order to accomplish transfection. Asused herein, the term “transfection” includes a variety of techniquesfor introducing an exogenous nucleic acid into a cell including calciumphosphate or calcium chloride precipitation, microinjection,DEAE-dextrin-mediated transfection, lipofection or electrophoration, allof which are routine in the art.

[0130] Transfected primary or secondary cells undergo sufficient numberdoubling to produce either a clonal cell strain or a heterogeneous cellstrain of sufficient size to provide the therapeutic protein to anindividual in effective amounts. The number of required cells in atransfected clonal heterogeneous cell strain is variable and depends ona variety of factors, including but not limited to, the use of thetransfected cells, the functional level of the exogenous DNA in thetransfected cells, the site of implantation of the transfected cells(for example, the number of cells that can be used is limited by theanatomical site of implantation), and the age, surface area, andclinical condition of the patient.

[0131] The transfected cells, e.g., cells produced as described herein,can be introduced into an individual to whom the product is to bedelivered. Various routes of administration and various sites (e.g.,renal sub capsular, subcutaneous, central nervous system (includingintrathecal), intravascular, intrahepatic, intrasplanchnic,intraperitoneal (including intraomental), intramuscularly implantation)can be used. One implanted in individual, the transfected cells producethe product encoded by the heterologous DNA or are affected by theheterologous DNA itself. For example, an individual who suffers fromfibrosis is a candidate for implantation of cells producing anantagonist of a component of the VEGF signaling pathway describedherein.

[0132] An immunosuppressive agent e.g., drug, or antibody, can beadministered to a subject at a dosage sufficient to achieve the desiredtherapeutic effect (e.g., inhibition of rejection of the cells). Dosageranges for immunosuppressive drugs are known in the art. See, e.g.,Freed et al. (1992) N. Engl. J. Med. 327:1549; Spencer et al. (1992) N.Engl. J. Med. 327:1541′ Widner et al. (1992) n. Engl. J. Med. 327:1556).Dosage values may vary according to factors such as the disease state,age, sex, and weight of the individual.

EXAMPLE Example 1

[0133] VEGF modulates CTGF mRNA Expression

[0134] The effects of VEGF on the expression of CTGF mRNA were studiedby Northern blot analysis in BREC and BRPC. 25 ng/ml VEGF increased CTGFmRNA (˜2.4 kb) levels in a time-dependent manner, reaching a maximumafter 6 h in BREC (3.1±0.70-fold, p<0.001) and after 9 h in BRPC(2.0±0.22-fold, p<0.01).

[0135] The dose response to VEGF-induced CTGF mRNA expression wasstudied after 6 h of VEGF stimulation. The expression of CTGF mRNA wasup-regulated in a dose-dependent manner, with significant increasesobserved at concentrations as low as 0.25 ng/ml in both BREC and BRPC.Maximal increases were observed at VEGF concentrations of 25 ng/ml inboth BREC and BRPC.

[0136] Since BREC and BRPC may express both KDR and Flt1, we examinedthe effects of P1GF, a Flt1-specific ligand, on the induction of CTGFgene expression in vascular cells. CTGF mRNA levels were not affectedafter stimulation of 25 ng/ml of P1GF in BREC. In contrast, P1GFincreased CTGF mRNA after 3 h of stimulation, which peaked after 9 h inBRPC (1.9±0.30-fold, p<0.01), suggesting that VEGF-induced CTGF geneexpression was mediated primarily by KDR in BREC and Flt1 in BRPC.

Example 2

[0137] VEGF Induction of CTGF Protein Production

[0138] To determine if the effects of VEGF on CTGF mRNA were correlatedwith its protein level, CTGF protein expression was assessed by Westernblot analysis using anti-human CTGF antibody. The detected size of CTGFprotein was ˜38 kDa in both BREC and BRPC. VEGF (25 ng/ml) increased thelevel of CTGF protein after 10 h in both BREC and BRPC. Comparativestudies were performed on the effects of VEGF (25 ng/ml) and TGF-β1 (10ng/ml) on the expression of CTGF mRNA and protein. VEGF and TGF-β1increased CTGF protein expression by a similar amount (2.5±0.4- and2.8±0.8-fold, respectively, in BREC). CTGF mRNA levels were alsoincreased a similar extent (3.0±0.3- and 3.3±0.5-fold, respectively).

Example 3

[0139] Effects of VEGF on the Half-life of CTGF mRNA

[0140] The effects of VEGF on the stability of CTGF mRNA were examined.Northern blot analyses were performed with addition of actinomycin D (5μg/ml) after 6 h of VEGF (25 ng/ml) stimulation. In BREC and BRPC3, thehalf-life of CTGF mRNA was 1.7 and 3.6 h, respectively. There was nosignificant difference between VEGF-treated and -untreated cells.

Example 4

[0141] Effects of Cycloheximide on CTGF mRNA Regulation

[0142] In order to examine the possibility that VEGF regulates CTGF mRNAexpression through new protein synthesis of cytokines or transcriptionfactors, cells were treated for 6 h with VEGF (25 ng/ml) and a proteinsynthesis inhibitor, cycloheximide (10 μg/ml). Cycloheximide did notprevent the increase of CTGF mRNA. Addition of both VEGF andcycloheximide increased CTGF mRNA 2.4±0.41-fold in BREC and2.5±0.40-fold in BRPC after 6 h as compared with cycloheximide alone(p<0.01). These data suggest that the stimulation of CTGF mRNAexpression by VEGF was not induced by increased synthesis of aregulatory protein.

Example 5

[0143] Involvement of Erk and PI3-Kinase-Akt in VEGF Signaling

[0144] Since Erk and PI3-kinase-Akt pathways have been reported to playcentral roles in VEGF signaling and biological actions, it wasinvestigated whether or not VEGF can activate Erk and PI3-kinase-Aktpathways equally in BREC and BRPC. Immunoblot analysis ofimmunoprecipitates of KDR from BREC stimulated with VEGF or P1GF usingan antibody to phosphotyrosine and PI3-kinase p85 subunit demonstratedthat VEGF, but not P1GF, promoted the tyrosine phosphorylation of KDRand interactions of KDR and p85 subunit of PI3-kinase. In contrast,Immunoblot analysis of immunoprecipitates of Flt1 from BRPC stimulatedwith VEGF or P1GF demonstrated that both VEGF and P1GF increased thetyrosine phosphorylation of Flt1 and interactions of Flt1 and p85subunit of PI3-kinase. These data suggest that VEGF can activate thereceptor tyrosine phosphorylation and interaction with PI3-kinase p85subunit in both KDR and Flt1.

[0145] To investigate the activation of Akt and Erk, we next performedimmunoblot analysis with anti-phosphorylated Akt or anti-phosphorylatedErk antibodies using total cell lysates from BREC or BRPC stimulatedwith VEGF. VEGF induced phosphorylation of both Akt and Erk in BREC by3.1- and 5.8-fold, but only induced phosphorylation of Akt in BRPC by2.6-fold. No effect on Erk phosphorylation was observed in BRPC. Thesedata suggest that VEGF activated both Erk and PI3-kinase-Akt pathways inBREC, but stimulated only PI3-kinase-Akt pathway in BRPC.

[0146] Since the activation of PI3-kinase by VEGF has not been reportedin BRPC, we studied the effects of VEGF on PI3-kinase activity in BRPC.The addition of VEGF (25 ng/ml) increased PI3-kinase activity in atime-dependent manner by 2.1±0.27-fold (p<0.01) after 5 min and by1.6±0.17-fold (p<0.05) after 10 min in BRPC.

Example 6

[0147] Effects of PKC, Erk, and PI3-Kinase Inhibition on VEGF-inducedCTGF Expression

[0148] To investigate the signaling pathways involved in VEGF-inducedCTGF expression, the effects of inhibition of PKC, Erk, and PI3-kinasewere determined. Cells were treated with 25 ng/ml VEGF for 6 h afterpretreatment with the kinase inhibitor GF 109203X, a classical and novelPKC-specific inhibitor (1 μM); PD98059, a MAPK/Erk kinase inhibitor (20μM); or wortmannin, a PI3-kinase inhibitor (100 nM). Neither GF 109203Xnor PD98059 had significant effects on VEGF-induced CTGF mRNAexpression, but wortmannin inhibited the effects of VEGF by 88±6.5%(p<0.01) in BREC and 78±22% (p<0.01) in BRPC. To confirm further theinvolvement of PI3-kinase in VEGF-induced CTGF expression, recombinantadenoviruses were used encoding dominant negative K-ras (DNRas),dominant negative extracellular signal-regulated kinase (DNErk), or Δp85of PI3-kinase. BREC were transfected with each adenoviral vector,followed by stimulation with 25 ng/ml VEGF for 6 h. Neither DNRas norDNErk had significant effects on VEGF-induced increase in CTGF mRNA, butΔp85 of PI3-kinase completely inhibited VEGF-induced CTGF expression(p<0.001).

Example 7

[0149] Role of PKCζ and Akt/PKB in VEGF-induced CTGF Expression

[0150] Since it has been reported that atypical PKC and Akt/PKB havesignificant roles as signaling molecules downstream of PI3-kinase, theinvolvement of PKCζ and Akt in this process were examined. BREC wereinfected with each adenoviral vector, followed by stimulation with 25ng/ml VEGF for 6 h. Neither wild type PKCζ nor dominant negative PKCζ(DNPKCζ) had significant effects on VEGF-induced increase in CTGF mRNA.In contrast, infection with constitutive active Akt (CAAkt) increasedCTGF mRNA expression 2.1±0.21-fold (p<0.01) without VEGF and2.5±0.40-fold with VEGF. Overexpression with adenoviral vectorcontaining dominant negative Akt (DNAkt) inhibited VEGF-induced CTGFexpression by 85±13%(p<0.01).

Example 8

[0151] Methods

[0152] A) Materials—Endothelial cell basal medium was purchased fromClonetics (San Diego, Calif.). Endothelial cell growth factor waspurchased from Roche Molecular Biochemicals. Dulbecco's modified Eagle'smedium and fetal bovine serum were obtained from Life Technologies, Inc.VEGF, placenta growth factor (P1GF), TGF-β1, and anti-CTGF antibody wereordered from R & D Systems (Minneapolis, Minn.). Anti-KDR (Flk1) andanti-Flt1 antibodies were purchased from Santa Cruz Biotechnology (SantaCruz, Calif.). Protein A-Sepharose was purchased from Amersham PharmaciaBiotech. Anti-phospho-Erk, anti-Erk, anti-phospho-Akt, and anti-Akt werepurchased from New England Biolabs (Beverly, Mass.). Anti-p85 andanti-phosphotyrosine were purchased from Upstate Biotechnology, Inc.(Lake Placid, N.Y.). Phosphatidylinositol (PI) was purchased from Avanti(Alabaster, Ala.), and PD98059, wortmannin, and GF 109203X were obtainedfrom Calbiochem. All other materials were ordered from Fisher and Sigma.

[0153] B) Cell Culture—Primary cultures of bovine retinal endothelialcells (BREC) and pericytes (BRPC) were isolated by homogenization and aseries of filtration steps as described previously. BREC weresubsequently cultured with endothelial cell basal medium supplementedwith 10% plasma-derived horse serum, 50 mg/liter heparin, and 50 μg/mlendothelial cell growth factor. BRPC were cultured in Dulbecco'smodified Eagle's medium with 5.5 mM glucose and 20% fetal bovine serum.Cells were cultured in 5% CO₂ at 37° C., and media were changed every 3days. Cells were characterized for their homogeneity by immunoreactivitywith anti-factor VIII antibody for BREC and with monoclonal antibody 3G5for BRPC. Cells remained morphologically unchanged under theseconditions, as confirmed by light microscopy. Only cells from passages 2through 7 were used for the experiments.

[0154] C) Recombinant Adenoviruses—cDNA of constitutive active Akt(CAAkt, Gag protein fused to N-terminal of wild type Akt) wasconstructed as in Burgering et al. (1995) Nature 376, 599-602. cDNA ofdominant negative Akt (DNAkt, substituted Thr-308 to Ala and Ser-473 toAla) was constructed as described in Kitamura et al. (1998) Mol. Cell.Biol. 18, 3708-3717. cDNA of dominant negative K-Ras (DNRas, substitutedSer-17 to Asn) was kindly provided by Dr. Takai (Osaka University). cDNAof dominant negative extracellular signal-regulated kinase (DNErk,substituted Lys-52 to Arg in ATP-binding site) was constructed asdescribed in Her et al. (1993) Biochem. J. 296, 25-3. cDNA of Δp85 waskindly provided by Dr. Kasuga(Kobe University). cDNA of PKCζ was kindlyprovided by Dr. Douglas Ways (Lilly). cDNA of dominant negative PKCζ(DNPKCζ, substituted Lys-273 to Trp in ATP-binding site) was constructedas in Uberall et al. (1999) J. Cell Biol. 144, 413-425

[0155] The recombinant adenoviruses were constructed by homologousrecombination between the parental virus genome and the expressioncosmid cassette or shuttle vector. The adenoviruses were applied at aconcentration of 1×10⁸ plaque-forming units/ml, and adenoviruses withthe same parental genome carrying the lacZ gene or enhanced greenfluorescein protein gene (CLONTECH, Palo Alto, Calif.) were used ascontrols. Expression of each recombinant protein was confirmed byWestern blot analysis and increased about 10-fold compared with cellsinfected with the control adenovirus.

[0156] D) Immunoprecipitation—Cells were washed three times with coldphosphate-buffered saline and solubilized in 200 μl of lysis buffer (1%Triton X-100, 50 mmol/liter HEPES, 10 mmol/liter EDTA, 10 mmol/litersodium pyrophosphate, 100 mmol/liter sodium fluoride, 1 mmol/litersodium orthovanadate, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 2mmol/liter phenylmethylsulfonyl fluoride). After centrifugation at12,000 rpm for 10 min, 1.0 mg of protein was subjected toimmunoprecipitation. To clear the protein extract, protein A-Sepharose(20 μl of a 50% suspension) was added to the cell lysates, after whichthey were incubated for 1 h, followed by centrifugation and collectionof the supernatant. A specific rabbit anti-KDR or Flt1 antibody wasadded and rocked at 4° C. for 2 h; 20 μl of protein A-Sepharose was thenadded, and the sample was rocked for another 2 h at 4° C. Fordenaturation, protein A-Sepharose antigen-antibody conjugates wereseparated by centrifugation, washed five times, and boiled for 3 min inLaemmli sample buffer.

[0157] E) Western Blot Analysis—Immunoprecipitated proteins or 30 μg oftotal cell lysates were subjected to SDS-gel electrophoresis andelectrotransferred to nitrocellulose membrane (Bio-Rad). The membranewas soaked in blocking buffer (phosphate-buffered saline containing 0.1%Tween 20 and 5% bovine serum albumin) for 1 h at room temperature andincubated with primary antibody overnight at 4° C. followed byincubation with horseradish peroxidase-conjugated secondary antibody(Amersham Pharmacia Biotech). Visualization was performed using theenhanced chemiluminescence detection system (ECL, Amersham PharmaciaBiotech) per the manufacturer's instructions.

[0158] F) PI3-Kinase Assay—PI3-kinase activities were measured by the invitro phosphorylation of PI as in Xia et al. (1996) J. Clin. Invest. 98,2018-2026. Cells were lysed in ice-cold lysis buffer containing 50 mMHEPES, pH 7.5, 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 2 mM Na₃VO₄, 10 mMNaF, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonylfluoride, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 μg/ml pepstatin.Insoluble material was removed by centrifugation at 15,000×g for 10 minat 4° C. PI3-kinase was immunoprecipitated from aliquots of thesupernatant with antiphosphotyrosine antibodies. After successivewashings, the pellets were resuspended in 50 μl of 10 mM Tris, pH 7.5,100 mM NaCl, and 1 mM EDTA. 10 μl of 100 mM MgCl₂ and 10 μl of PI (2μg/μl) sonicated in 10 mM Tris, pH 7.5, with 1 mM EGTA was added to eachpellet. The PI3-kinase reaction was initiated by the addition of 5 μl of0.5 mM ATP containing 30 μCi of [γ-³²P]ATP. After 10 min at roomtemperature with constant shaking, the reaction was stopped by theaddition of 20 μl of 8 N HCl and 160 μl of chloroform/methanol (1:1).The samples were centrifuged, and the organic phase was removed andapplied to silica gel TLC plates developing in CHCl₃/CH₃OH/H₂O/NH₄OH(60:47:11:2). The radioactivity in spots was quantified byPhosphorlmager (Molecular Dynamics, Sunnyvale, Calif.).

[0159] G) Amplification of Human CTGF cDNA Using ReverseTranscriptase-Polymerase Chain Reaction (PCR)—cDNA templates for PCRwere synthesized by reverse transcriptase (First Strand cDNA SynthesisKit, Amersham Pharmacia Biotech) from human fibroblast according to themethod recommended by the manufacturer. A standard PCR was performed(PCR optimizer kit, Invitrogen, Carlsbad, Calif.) using5′-AGGGCCTCTTCTGTGACTTCG-3′ (sense primer) and5′-TCATGCCATGTCTCCGTACATC-3′ (antisense primer). The PCR products werethen subcloned into a vector (pCRII, Invitrogen) and sequenced in theirentirety, and comparison with the published human sequences revealedcomplete sequence identity. This cDNA probe was used for hybridization.

[0160] H) Northern Blot Analysis—Total RNA was isolated usingacid-guanidinium thiocyanate, and Northern blot analysis was performed.Total RNA (20 μg) was electrophoresed through 1% formaldehyde-agarosegels and then transferred to a nylon membrane. ³²P-Labeled cDNA probeswere generated by use of labeling kits (Megaprime DNA labeling systems,Amersham Pharmacia Biotech). After ultraviolet cross-linking using a UVcross-linker (Stratagene, La Jolla, Calif.), blots were pre-hybridized,hybridized, and washed in 0.5×SSC, 5% SDS at 65° C. with 4 changes over1 h. All signals were analyzed using a Phosphorlmager, and lane loadingdifferences were normalized.

[0161] I) Analysis of CTGF mRNA Half-life—CTGF mRNA half-lifeexperiments were carried out using BREC and BRPC. The cells were exposedto vehicle or VEGF (25 ng/ml) for the indicated periods prior to mRNAstability measurements. Transcription was inhibited by the addition ofactinomycin D (5 μg/ml). For inhibition of protein synthesis, cells weretreated with cycloheximide (10 μg/ml) for the times indicated.

[0162] J) Statistical Analysis—Determinations were performed intriplicate, and all experiments were repeated at least three times.Results are expressed as the mean±S.D., unless otherwise indicated.Statistical analysis employed Student's t test or analysis of varianceto compare quantitative data populations with normal distributions andequal variance. Data were analyzed using the Mann-Whitney rank sum testor the Kruskal-Wallis test for populations with non-normal distributionsor unequal variance. A p value of <0.05 was considered statisticallysignificant.

[0163] All patents and references cited herein are hereby incorporatedby reference in their entirety.

We claim:
 1. A method of decreasing fibrosis in a tissue of a subject,comprising: identifying a subject in need of decreased fibrosis; andadministering to the subject an agent that inhibits a component of theVEGF signal transduction pathway, wherein the agent decreases aconnective tissue growth factor (CTGF) activity in the tissue of thesubject.
 2. The method of claim 1, wherein the agent decreases the levelor activity of VEGF or a VEGF receptor (VEGFR).
 3. The method of claim2, wherein the VEGFR is KDR, Flt1, Flt4 or neuropilin.
 4. The method ofclaim 2, wherein the agent is selected from the group of: a VEGF bindingor VEGF receptor (VEGFR) binding protein that inhibits VEGF binding toVEGFR; an antibody to VEGF or VEGFR that inhibits VEGF or VEGFRactivity; a mutated VEGF or VEGFR or fragment thereof that inhibits VEGFsignaling; a VEGF or VEGFR nucleic acid molecule that inhibitsexpression of VEGF or VEGFR; and a small molecule that inhibitstranscription or activity of VEGF or VEGFR.
 5. The method of claim 1,wherein the agent decreases the level or activity of AKT.
 6. The methodof claim 5, wherein the agent is selected from the group of: an AKTbinding protein that inhibits AKT activity; an antibody to AKT thatinhibits AKT activity; a mutated AKT or fragment thereof that inhibitsAKT activity; an AKT nucleic acid molecule that inhibits expression ofAKT; and a small molecule that inhibits transcription or activity ofAKT.
 7. The method of claim 1, wherein the agent decreases the level oractivity of PI3 kinase.
 8. The method of claim 7, wherein the agent isselected from the group of: a PI3 kinase binding protein that inhibitsPI3 kinase activity; an antibody to PI3kinase that inhibits PI3 kinaseactivity; a mutated PI3 kinase or fragment thereof that inhibits PI3kinase activity; a PI3 kinase nucleic acid molecule that inhibitsexpression of PI3 kinase; and a small molecule that inhibitstranscription or activity of PI3 kinase.
 9. The method of claim 7,wherein the agent is LY294002.
 10. The method of claim 7, wherein theagent is wortmannin.
 11. The method of claim 1, wherein the subject hasa fibrosis related disorder.
 12. The method of claim 1, wherein thetissue is skin tissue, lung tissue, cardiac tissue, kidney tissue, livertissue, or retinal tissue.
 13. A method of decreasing angiogenesis in atissue of a subject, comprising: identifying a subject in need ofdecreased angiogenesis; and administering to the subject an agent thatinhibits a component of the VEGF signal transduction pathway, whereinthe agent decreases a connective tissue growth factor (CTGF) activity inthe tissue of the subject.
 14. The method of claim 13, wherein thetissue is skin tissue, lung tissue, cardiac tissue, kidney tissue, livertissue, retinal tissue, or cancerous or tumor tissue.
 15. The method ofclaim 13, wherein the agent is selected from the group of: a) an agentthat inhibits VEGF activity; b) an agent that inhibits VEGFR signaling;c) an agent that inhibits PI3 kinase activity; d) an agent that inhibitsa VEGFR interaction with p85 subunit of PI3-kinase; e) an agent thatinhibits AKT activity; and f) an agent that inhibits ERK activity. 16.The method of claim 15, wherein the VEGFR is KDR or Flt1.
 17. A methodof increasing fibrosis in a subject, comprising: identifying a subjectin need of increased fibrosis; and administering to the subject an agentthat induces a component of the VEGF signal transduction pathway,wherein the agent increases a connective tissue growth factor (CTGF)activity in the tissue of the subject.
 18. The method of claim 17,wherein the agent is selected from the group of: a) an agent thatdecreases a PKC activity; b) an agent that increases VEGF activity; c)an agent that increases VEGFR signaling; d) an agent that increasesVEGFR interaction with p85 subunit of PI3 -kinase; e) an agent thatincreases PI3 kinase activity; and f) an agent that increases AKTactivity.
 19. The method of claim 17, wherein the agent is VEGF orplacental growth factor (PlGF)
 20. The method of claim 17, wherein theagent is a transition metal ion.
 21. The method of claim 18, wherein theVEGFR is KDR or Flt1.
 22. A method of screening for a compound thatdecreases fibrosis or angiogenesis, comprising: providing a cell,tissue, or subject; contacting the cell, tissue, or subject with a testcompound; and determining whether the test compound inhibits a componentof the VEGF signaling pathway.
 23. The method of claim 22, furthercomprising contacting the cell, tissue, or subject with VEGF.
 24. Themethod of claim 22, further comprising assaying the cell, tissue orsubject for a CTGF activity.
 25. The method of claim 22, wherein thecell is an endothelial cell
 26. The method of claim 22, wherein the cellis a bovine retinal endothelial cell (BREC) or bovine retinal pericyte(BRPC).